Method of collecting highly pure polyhydroxyalkanoate from microbial cells

ABSTRACT

The present invention has an object to provide a method for separating and purifying a PHA without causing a serious decrease of the molecular weight to obtain a highly pure PHA in a high yield, which comprises efficiently removing cell components other than PHA particles from a cultured PHA-containing microbial cell. Another object of the present invention is to provide a method for obtaining an agglomerate of PHA particles. The method for recovering a PHA according to the present invention is a method which comprises efficiently disrupting a cell to recover the PHA by carrying out a physical disruption treatment and an alkali addition at low temperature for an aqueous suspension of the PHA-containing microbial cell, and then treating the PHA with an enzyme and/or a surfactant. Moreover, the particle diameter of the PHA may be enlarged by suspending the PHA in a hydrophilic solvent and/or water, and stirring at a temperature equal to or below the boiling point of said suspension, to agglomerate said PHA.

TECHNICAL FIELD

The present invention relates to a separation/recovery method of abiodegradable polyester resin from microbial cells, and to anagglomeration method of said resin particle.

BACKGROUND ART

Polyhydroxyalkanoates (hereinafter referred to briefly as “PHA” s) arethermoplastic polyesters which are synthesized and stored as an energystorage substance in cells of a variety of microorganisms. The PHAs,which are produced by microorganisms using natural organic acids or oilsas carbon sources, are completely biodegraded by a microorganism in soilor water to be taken up in the carbon cycle of the natural world.Therefore, PHAs can be said to be an environment-conscious plasticmaterial which hardly causes adverse effects for ecological system. Inthese years, a synthetic plastic came into a serious social problem inview of environment pollution, waste disposal and oil resource, thus thePHA has attracted attention as an eco-friendly green plastic and itspractical applications are longed for. Also in the field of medicaltreatment, it is considered possible to use PHAs as implant materialswhich do not require recovery, or vehicles for drug. Thus, practicalapplications thereof have been expected.

Since PHAs synthesized by a microorganism are stored in cells usually inthe form of granules, it is required a procedure for separating PHAsfrom microbial cells to utilize them as plastics. The known technologyfor separation and purification of PHAs from microbial cells can beroughly classified into technologies which comprise extracting a PHAfrom microbial cells with organic solvents capable of solving a PHA andtechnologies which comprise removing cell components other than PHAs bycell disruption or solubilization.

In earlier researches, many technologies for separating and purifyingPHAs by extraction using organic solvents were reported (see JapaneseKokai Publication Sho-55-118394, Japanese Kokai PublicationSho-57-65193, Japanese Kokai Publication Sho-63-198991, Japanese KokaiPublication Hei-02-69187 and Japanese Kokai Publication Hei-07-79788).In these reports, halogen compounds such as chloroform were used asorganic solvents having the highest solubility of PHAs, but when a PHAwas dissolved in such a solvent, viscosity of solution became very highand handling of the solution became difficult. Therefore, for extractinga PHA, it was needed to set the polymer concentration in a range asextremely low as about 2 to 3%, thus significantly large amount ofsolvent was required. In addition, for crystallizing a PHA from asolvent layer in a high yield, a large amount as 4 to 5 times as theabove solvent of poor solvents for a PHA, such as methanol and hexane,were separately required. Accordingly, for production on an industrialscale, large-scale equipment is required. Moreover, a PHA cannot beproduced in a low cost since these technologies require huge amount ofsolvent, and therefore it takes much cost for solvent recovery and costdue to solvent loss. Due to such reasons as mentioned above, thesemethods have not been put into practice.

On the other hand, various technologies have been reported whichcomprise solubilizing and removing cell components other than PHAs bychemical treatments or physical disruption treatments to recover PHAs inthe form of granules.

As a method for chemically treating a microbial cell (hereinafter,sometimes referred to as “cell”), J. Gen. Microbiology, 1958 vol. 19, p.198-209 discloses a technology which comprises treating a suspension ofa microbial cell with sodium hypochlorite and solubilizing cellcomponents other than a PHA to recover the PHA. In this technology,marked degradation of a PHA is caused in solubilizing the cellcomponents other than the PHA, and processing ways into products arelimited. Moreover, sensible smell of chlorine is left behind in PHAs,which is undesirable for a polymer product. Thus, this technology is notconsidered to be suitable for practical use. Japanese Kokoku PublicationHei-04-61638 discloses a recovering process which comprises heattreatment in combination with use of an enzyme and/or a surfactant. Inthis process, heating a suspension to 100° C. or above beforehand isrequired to decompose nucleic acids, since the suspension becomes highlyviscous by free nucleic acids when cells are dissolved by an enzymetreatment. However, the molecular weight of a PHA decreases markedly byheating to 100° C. or above, and an application to products will becomeimpossible. Moreover, despite this technology is very complicated andrequires many processes, purity of an obtained PHA is as much as about88% in general, and 97% even at the maximum. Additionally, a technologywhich comprises treating a PHA-containing microbial cell with asurfactant, decomposing a nucleic acid released from the cell withhydrogen peroxide at 80° C. for 3 hours, and separating a PHA with apurity of 99% (see Japanese Kohyo Publication Hei-08-502415), and atechnology which comprises heating a suspension of a PHA-containingmicroorganism to 50° C. or higher under a strongly acidic condition ofbelow pH 2, and separating a PHA (see Japanese Kokai PublicationHei-11-266891) have been proposed. Under these heating conditions, themolecular weight of the PHA decreases remarkably, therefore even if itspurity is improved, applications to products are still impossible.

On the other hand, as a method applying physical disruption treatments,a technology have been reported, which comprises carrying out an alkaliaddition with a high-pressure disruption or a combination of ahigh-pressure disruption. Although “Bioseparation”, 1991, vol. 2, p.95-105 does not describe purity or yield of a polymer, cell componentsremain in a poly-3-hydroxybutyrate (PHB) fraction and the purity of PHBis presumably not high since high-pressure disruption is carried outunder a condition where pH is returned to neutral after adding alkali toa cell suspension containing PHB. Japanese Kokai PublicationHei-07-31487 discloses a technology which comprises heating to 80° C.after an alkali addition to a cell suspension containing a PHA, stirringthe mixture for 1 hour and recovering a polymer by centrifugation;Japanese Kokai Publication Hei-07-31488a discloses a technology whichcomprises carrying out high-pressure disruption at 70° C.; and atechnology considered to develop the method described in the above“Bioseparation”, 1991, vol. 2, p. 95-105, that is a technology whichcomprises carrying out high-pressure disruption at 70° C. or higherafter the alkali addition in Japanese Kokai Publication Hei-07-31489,respectively. By these technologies, since the processes are carried outin high temperature conditions, there is a tendency toward remarkabledecrease in a molecular weight of a PHA in some conditions. Moreover,purity is also as low as about 66 to 85%, thus these technologies maynot be applied to actual industrial processes.

As mentioned above, we can find it very difficult to recover a PHA froma cultured cell without decreasing the molecular weight but with highpurity and a high yield in a low-cost on an industrial production.

By the way, when a PHA is obtained by a technology comprisingsolubilizing to remove cell components other than a PHA by a chemical orphysical treatment and recovering the PHA in the form of granules, theobtained PHA usually occurred as a form of fine particles havingdiameters of several microns. It is more difficult to separate such fineparticles from a liquid medium as compared with the case of particleshaving a larger diameter. Moreover, these fine particles are consideredto have problems such as a risk to cause dust explosion and/oraccumulation in lungs when aspirated, thus care should be taken forhandling.

In order to avoid these problems, there have been attempts to enlargethe particle diameter by agglomerating a PHA. For example, anagglomeration method by heating or an alkaline metal salt has beendeveloped. As a method of agglomeration by heating, a technology isdisclosed, which comprises agglomerating PHB by heating a PHB-containingsuspension to the vicinity of the melting point of PHB (180° C.) (seeBailey, Neil A.; George, Neil; Niranjan, K.; Varley, Julie. BiochemicalEngineering group, University Reading, “I Chem E Res. Event, Eur. Conf.Young Res. Chem. Eng.” (Britain), 2nd edition, Institution of ChemicalEngineers, 1996, vol. 1, p. 196-198). Moreover, Japanese KohyoPublication Hei-07-509131 discloses a technology to enlarge a particlediameter of a copolymer comprised of 3-hydroxybutyrate (3HB) and3-hydroxyvalerate (3HV) (hereinafter such copolymer is referred to as“PHBV”), which comprises injecting steam with an appropriate temperatureand pressure directly into an aqueous suspension of PHBV, and heating-and stirring the suspension at 120 to 160° C. However, they are notpractical since these technologies require heating at a hightemperature, the molecular weight of PHA decreases remarkably, andfurther special equipment with a pressure resistance is required.Alternatively, as a method for agglomerating a PHA by adding an alkalinemetal salt, a technology for agglomeration using a divalent cation (seeJ. Biotechnol., 1998, vol. 65 (2, 3), p. 173-182, and Japanese KohyoPublication Hei-05-0507410) has been disclosed. However, thesetechnologies are not preferable in, for example, that polymeragglomeration strength is not always high, that a metal salt iscontaminated into a polymer, and the like ploblems. Alternatively, atechnology which comprises agglomerating PHB by blowing ultrafinebubbles into a PHB suspension to raise a flock to the surface (see Spec.Publ. -R. Soc. Chem., 1994, vol. 158 (Separations for Biotechnology 3),p. 113-119) has also been reported. However, the agglomerate obtained bythis technology has a diameter of 2 to 45 μm, and cannot be said as asufficient size.

Thus, any methods for controlling a molecular-weight decrease of a PHAand carrying out agglomeration effectively have not been known in thestate of the art.

As described above, there lies a large obstacle for practicalapplications in studies of PHAs, which are one species of biodegradablepolymers derived from microorganisms, since any processes with low costsand suitable for industrial productions have not been establishedrespectively in recovering PHAs from microbial cells, and further inagglomerating PHA particles carried out according to need.

SUMMARY OF THE INVENTION

As mentioned above, in the process of recovering a PHA from a microbialcell, conventional methods cannot be said as processes with low costsand suitable for industrial productions. Moreover, the present inventorshave carried out a preliminary investigation, and as a result, theyfound that such conventional methods, which require a chemical treatmentby hypochlorous acid, hydrogen peroxide, acid, a large amount of alkali,etc., and a reaction under a high temperature, could hardly be utilized.Especially in the case that a PHA composed two or more species ofmonomer components was used, the molecular weight tends to decrease moreremarkably than the case of using a homopolymer PHB.

Therefore, the object of the present invention is to solve theabove-mentioned problems in the conventional methods, and to provide amethod for separating and purifying a PHA through fewer steps withoutcausing a serious decrease of the molecular weight to obtain a highlypure PHA in a high yield, which comprises efficiently removing cellcomponents other than PHA particles from a cultured PHA-containingmicrobial cell. Another object of the present invention is to provide amethod for obtaining an agglomerate of PHA particles.

The present inventors carried out intensive investigations on anindustrially advantageous recovery method of a PHA from a microbialcell. As a result thereof, the inventors found it possible toefficiently recover highly pure PHA by producing a PHA using amicroorganism, adding an alkali to an aqueous suspension of themicrobial cell containing a PHA while stirring and carrying out physicaldisruption treatment at comparatively low temperature, then recoveringthe PHA, treating the PHA with an enzyme and/or surfactant in an aqueoussuspension or wet state, and further washing said PHA with a hydrophilicsolvent and/or water. Furthermore, the inventors also found it possibleto enlarge a particle diameter of a PHA by suspending the PHA in ahydrophilic solvent and/or water and agglomerating them by stirring at atemperature equal to or below the boiling point of said suspension. Bythese methods, the inventors have succeeded in preventing the molecularweight decrease, which has been a very difficult subject up to present,and in recovering a PHA having purity of 99% or more in a yield of 90%or more. By further agglomeration, the inventors have completed theproduction method of a PHA capable of avoiding difficulty of handlingand/or the risk of dust explosion. By the completion of the presentinvention, practical applications of a biodegradable polymer derivedfrom a microbial cell will become possible.

That is, the present invention relates to a method for recovering a PHAfrom a PHA-containing microbial cell

which comprises;

(a) a step comprising adding an alkali to an aqueous suspension of thePHA-containing microbial cell while stirring and carrying out a physicaldisruption treatment to disrupt the cell, solubilizing or emulsifyingcell substances other than the PHA in said cell, and then separating thePHA from the aqueous suspension, and

(b) a step comprising treating the separated PHA with an enzyme and/or asurfactant to solubilize impurities adhering to the PHA or to solubilizethem after decomposing, and then washing the PHA with a hydrophilicsolvent and/or water.

Moreover, the present invention relates to the above-mentioned methodfor recovering a PHA

which further comprises;

(c) a step comprising suspending the washed PHA in a hydrophilic solventand/or water and stirring at a temperature equal to or below the boilingpoint of said suspension and agglomerating the PHA to enlarge theparticle diameter thereof, and then separating the agglomerated PHA fromthe suspension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents an explanatory drawing showing a microbial celldisruption equipment used for separating and purifying apoly-3-hydroxyalkanoic acid according to the present invention.

EXPLANATION OF NUMERALS

1 Microbial cell disruption equipment

2 Stirring equipment

3 pH Detection and control equipment

4 Pump

5 Pipeline

6 pH Control agent storage tank

7 pH Indicator

8 Pipeline

9 Disruption equipment

10 Pump

-   11 Cell disruption tank

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in further detailwith a preferable embodiment.

The method for recovering polyhydroxyalkanoate according to the presentinvention comprises the following steps (a) and (b);

(a) a step comprising adding an alkali to an aqueous suspension of apolyhydroxyalkanoate-containing microbial cell while stirring andcarrying out a physical disruption treatment to disrupt the cell, andsolubilizing or emulsifying cell substances other than thepolyhydroxyalkanoate in said cell, and then separating thepolyhydroxyalkanoate from the aqueous suspension, and

(b) a step comprising treating the separated polyhydroxyalkanoate withan enzyme and/or a surfactant to solubilize impurities adhering to thepolyhydroxyalkanoate or to solubilize them after decomposing, and thenwashing the polyhydroxyalkanoate with a hydrophilic solvent and/orwater.

Firstly, a polyhydroxyalkanoate (PHA) as used in this specification is ageneric term meaning any or all polymers composed of hydroxyalkanoates.The hydroxyalkanoate components are not particularly restricted, butspecifically there may be mentioned, for example, 3-hydroxybutyrate(3HB), 3-hydroxyvalerate (3HV), 3-hydroxypropionate, 4-hydroxybutyrate,4-hydroxyvalerate, 5-hydroxyvalerate, 3-hydroxypentanoate,3-hydroxyhexanoate (3HH), 3-hydroxyheptanoate, 3-hydroxyoctanoate,3-hydroxynonanoate, 3-hydroxydecanoate, etc.

The PHA of the present invention may be a homopolymer of one of thesehydroxyalkanoates or a copolymer obtainable by copolymerizing two ormore species of these. Particularly, the recovering method of thepresent invention is suitable since the molecular weight hardlydecreases, as described below, in the case of using a copolymer, whosemolecular weight tends to be decreased in a conventional method.

As specific examples of the PHA, there may be mentioned PHB (ahomopolymer of 3HB), PHBV (a binary copolymer composed of 3HB and 3HV),PHBH (a binary copolymer composed of 3HB and 3HH, see Japanese PatentPublication No. 2777757), PHBHV (aternary copolymer composed of 3HB, 3HVand 3HH, see Japanese Patent Publication No. 2777757), etc. Particularlyamong them, a copolymer comprising 3HH as a monomer unit is preferablesince it has degradability as a biodegradable polymer and softness, andmore preferably PHBH.

In the case of PHBH, The compositional ratio of monomer unitsconstituting PHBH is not particularly restricted but ones containing 1to 99mol % of 3HH unit are preferred and ones containing 3 to 30% of 3HHare more preferred since they show preferable workability. Moreover, inthe case of PHBHV, the compositional ratio of monomer units constitutingof PHBHV is not particularly restricted, but ones containing 1 to 95 mol% of 3HB unit, 1 to 96 mol % of 3HV unit, and 1 to 30 mol % of 3HH unitare preferred.

From a practical point of view, it is preferred that the PHA has theaverage molecular weight determined by a gel chromatography method, inwhich polystyrene is set as a molecular weight standard, of 10,000 ormore. It is more preferred 50,000 or more, still more preferably 100,000or more, and particularly preferably 200,000 or more.

The microorganism to be used in the present invention is notparticularly restricted provided that it is a microorganism capable ofstoring a PHA in cells. For example, there may be mentionedmicroorganisms belonging to the genus Aeromonas, Alcaligenes,Azotobacter, Bacillus, Clostridium, Halobacterium, Nocardia,Rhodospirillum, Pseudomonas, Ralstonia, Zoogloea, etc. Morespecifically, there may be mentioned Aeromonas caviae, etc. as amicroorganism belonging to the genus Aeromonas, Alcaligenes lipolytica,Alcaligenes latus, etc. as ones belonging to the genus Alcaligenes, andRalstonia eutropha, etc. as a microorganism belonging to the genusRalstonia, for instance.

These microorganisms can store a PHA in cells by controlling cultureconditions.

Alternatively, a transformant transformed with a gene group involving aPHA synthesis may also be used as these microorganisms. In that case,the host is not particularly restricted, and there may be mentionedmicroorganisms such as Escherichia coli (the genus Escherichia) andyeast belonging to the genus Candida, Saccharomyces, Yarrowia, etc. (WO0188144), in addition to the above-mentioned microorganisms.

Among the above microorganisms to be used in the present invention,Aeromonas caviae belonging to the genus Aeromonas and the transformanttransformed with a PHA synthase group gene derived from said Aeromonascaviae are preferable since they have an excellent synthesizing abilityof PHBH. In particular, more preferred is a transformant obtained byintroducing a PHA synthase group gene derived from Aeromonas caviae intoRalstonia eutropha.

As one example of said microorganisms, Ralstonia eutrophaPHB-4/pJRDEE32d13 strain obtained by introducing a PHA synthase groupgene derived from Aeromonas caviae into Ralstonia eutropha maypreferably be used. Said Ralstonia eutropha PHB-4/pJRDEE32d13 strain isinternationally deposited based on Budapest Treaty to National Instituteof Advanced Industrial Science and Technology International PatentOrganism Depositary, Tsukuba, Central 6, 1-1-1 Higashi, Ibaraki, Japanon Aug. 7, 1997 (Heisei-9), with an accession No. FERM BP-6038 under thename of Alcaligenes eutrophus AC32.

In the practice of the present invention, microbial cells obtained byculturing microorganisms mentioned above in a suitable condition tostore a PHA therein, is utilized. The above culturing method is notparticularly restricted but the method known to the person skilled inthe art, for example the method described in Japanese Kokai Publication2001-340078, can be applied.

In recovering a PHA, it is naturally preferable that the PHA content inthe cultured microbial cell is higher. In the application for anindustrial production, PHA content in dried cells is preferably 50weight % or more. Taking subsequent separation operations and purity ofa separated polymer into consideration, the PHA content in dried cellsis more preferably 60 weight % or more, and still more preferably 70weight % or more.

Although it is possible to directly proceed to the step (a) aftercompletion of the culture, it is also possible to proceed to the step(a) after recovering cells by methods such as centrifugation andmembrane separation, which are known to the person skilled in the art,or recovering cells after killing cells by heating and the likeprocedure. The temperature for the heating is preferably 50° C. to 70°C.

In the step (a) of the present invention, it is important to add analkali to an aqueous suspension of a PHA-containing microbial cell whilestirring and carrying out physical disruption treatment of the aqueoussuspension. That is, a process is actually carried out, which comprises(1) preparing an aqueous suspension of the PHA-containing microbialcell, (2) starting physical disruption treatment while stirring saidaqueous suspension, and then (3) adding an alkali while continuingstirring and physical disruption.

When an alkali is added to the suspension of a PHA-containing microbialcell without carrying out the physical disruption, nucleic acids, cellwalls, cell membranes, insoluble proteins, etc. are flowed out from themicrobial cell together with a PHA. At this time, the present inventorsfound that viscosity of the suspension significantly rose, and evenstirring of the suspension became impossible in some conditions, therebyrecovery of the PHA became impossible. Additionally, the presentinventors found that a PHA was easily decomposed in recovering the PHAwhen a physical disruption (for example, cell disruption andemulsification by a high-pressure homogenizer) after an alkali additionto make pH level of the suspension to be 10 or higher. On the contrary,they unexpectedly found that a PHA was hardly decomposed when thephysical disruption was carried out before the alkali addition.

Accordingly, in the present invention, it becomes possible to easilyseparate and recover a PHA from a suspension with inhibitingdecomposition of the PHA by, in the step (a), once starting a physicaldisruption, and then gradually adding an alkali while continuing thephysical disruption to promote solubilization or emulsification of theinsoluble substances (cell substances) other than the PHA.

The aqueous suspension containing a PHA-containing microbial cell usedin the step (a) means a suspension prepared by suspending thePHA-containing microbial cell obtained as above in water.

A suspension concentration of said microbial cell is preferably 500 g/Lor less in terms of dried cell weight per liter of the aqueoussuspension, and in view of stirring easiness of the suspension ofmicrobial cell, more preferably 300 g/L or less. The lower limit ispreferably 80 g/L or more.

The stirring means of the above aqueous suspension is not particularlyrestricted, but an emulsification-dispersion machine or a sonicationdisruption machine is preferably used for stirring to efficientlydiffuse an alkali to be added and efficiently disrupting high-viscosityDNAs flowed out from the cell. More preferred is theemulsification-dispersion machine, and for example, SILVERSONMIXERmanufactured by Silverson Machines, Inc., England, CLEAR MIXmanufactured by M-TECHNIQUE, Japan, and Ebara Milder manufactured byEbara Corporation, Japan, etc. may be used, but is not limited to these.

In the present invention, equipment for carrying out the physicaldisruption treatment is not particularly restricted, but there maybementioned a high-pressure homogenizer, a sonication disruption machine,an emulsification-dispersion machine, a bead mill, etc. Among them,preferred is a high-pressure homogenizer, and more preferred are onesbelonging to a type in which an aqueous suspension of the polymer isintroduced into a pressure-resistant container having a micro-opening,and the suspension is pushed out from the opening by applying highpressure. As such equipment comprising a pressure-resistant containerand a pressurization mechanism, for example, a high-pressure homogenizermanufactured by Italy Niro Soavi S.p.A is preferably used. Moreover,such equipment includes Bran + Luebbe continuous cell disruptioner(product of Bran + Luebbe GmbH, Germany), and Microfluidizer (product ofMicrofluidics, U.S.), etc., but it is not limited to these.

In such high-pressure homogenizers, since large shearing force isapplied to a microbial cell, the microbial cell is efficiently destroyedand separation of a polymer is promoted. Moreover, in such equipment,since a high pressure is applied to the opening and becomes hightemperature instantaneously, it is preferable to cool the microbialcell-containing suspension in a general cooling bath circulator,according to need, to prevent the temperature elevation and carry out adisruption treatment at 20 to 40° C. The molecular weight of a PHAhardly decreases when the treatment is carried out at such acomparatively low temperature. Therefore, in the preferable embodimentof the present invention, it is preferred to utilize a processcomprising adding an alkali while carrying out a physical disruption at20 to 40° C.

The alkali to be used in the step (a) is not particularly restrictedprovided that it is capable of disrupting a cell wall of thePHA-containing microorganism and discharging a PHA from inside tooutside of the cell. The alkali includes, but not limited to, forexample, alkali metal hydroxides such as sodium hydroxide, potassiumhydroxide and lithium hydroxide; alkali metal carbonates such as sodiumcarbonate and potassium carbonate; alkali metal hydrogen carbonates suchas sodium hydrogen carbonate and potassium hydrogencarbonate; alkalimetal salts of organic acids such as sodium acetate and potassiumacetate; alkali metal borates such as borax; alkali metal phosphatessuch as trisodium phosphate, disodium hydrogenphosphate, tripotassiumphosphate and dipotassium hydrogenphosphate; alkaline earth metalhydroxides such as barium hydroxide; aqueous ammonia, etc. These may beused alone or two or more of them may be used in combination. Amongthese, alkali metal hydroxides and alkali metal carbonates are preferredsince they are suitable for an industrial production and the costs arereasonable. And more preferred are sodium hydroxide, potassiumhydroxide, lithium hydroxide, sodium carbonate and the like.

In the step (a) of the present invention, it is preferable to controlthe pH level during the addition of an alkali. The preferable pH rangefor efficiently solubilizing insolubilities (cell substances) derivedfrom cells other than a PHA and having no adverse effect to the PHAitself is pH 9 to 13.5, and more preferably pH 10 to 13. If the pH levelis higher than 13.5, molecular weight of a PHA tends to decrease, and ifthe pH level is below 9, the disruption effect tends to be reduced.

Therefore, a method may be preferably used, which comprises adding analkali continuously or intermittently to a suspension of microbial cellswhile controlling the pH within the desired levels. In the presentinvention, controlling the pH in such manner prevents pH level from toomuch elevating, which will occur in the case of adding the whole alkaliat once. Furthermore, constantly maintaining the alkali condition to bemore than some extent makes it possible to maintain insoluble proteinsto be a solubilizable state, and it becomes unnecessary to heat thesuspension at a high temperature. As a result, the molecular weightdecrease of a PHA may be prevented more efficiently.

The temperature on carrying out the step (a) is preferably from 10 to45° C., and more preferably from 20 to 40° C. in view of preventing themolecular weight decrease of a PHA more efficiently.

As mentioned above, when a physical disruption such as high-pressuredisruption is carried out while maintaining the pH to be an arbitrarylevel within 9 to 13.5 in the step (a), a treatment at such a lowtemperature as 20 to 40° C. becomes possible, and the molecular weightdecrease might be suppressed to 10% or less even in the case of PHBH.Namely, it is particularly preferred to carry out the physicaldisruption in a pH level of 9 to 13.5, at 20 to 40° C. When themicrobial cells are disrupted under such preferable alkali condition,more reproducible result may be obtained.

The separation of a PHA from the suspension may be carried out by, forexample, centrifugation, membrane separation, filter filtration, etc.

In the followings, the step (a) is explained in further detail by usingFIG. 1, which represents a schematic diagram showing preferableequipment for carrying out the step (a). Of course, the presentinvention is not limited to these equipment examples.

The reference numeral 1 in FIG. 1 indicates a microbial cell disruptionequipment according to the invention as a whole. The reference numeral 6indicates a pH control agent storage tank for reserving an alkali agent,and the agent in this pH control agent storage tank 6 is fed by a pump 4to the cell disruption tank 11 through a pipeline 5 to adjust the pH ofa microbial cell suspension in the cell disruption tank 11 according toneed. This cell disruption tank 11 is equipped with a stirring means 2for uniformly stirring and mixing the pH control agent, which is fedfrom the pH control agent storage tank 6, with the microbial cellsuspension in the cell disruption tank 11. The cell disruption tank 11is further equipped with a pH detection-control means composed of a pHmeter 7 and a pH sensor-controller 3 for detecting the pH of themicrobial cell suspension in the cell disruption tank 11 and controllingthe rate of feed of the pH control agent by said pump 4 so that apredetermined pH level may be established. The cell disruption tank 11works also as a cooling bath circulator, and the microbial cellsuspension may be maintained at the desired constant temperature.

Referring to FIG. 1, the microbial cell suspension in the celldisruption tank 11 is fed via a pump 10 to a disruption equipment 9,where nucleic acids, which may be causative of viscosity elevation, isefficiently disrupted, and resultant mixture is fed to the celldisruption tank 11 via a pipeline 8. The added alkali is immediatelydiffused by the stirring means 2, and the microbial cell suspension ishomogenized, thus making it possible to strictly control the pH level ofthe cell suspension. At this point, it is preferred to stir sufficientlyin order to prevent the alkali concentration from becoming partiallyhigh, and a polymer would not be subjected to hydrolysis. Thefluctuation range of the pH level to be controlled is preferably within±1 of the set value, and more preferably within ±0.5. And it ispreferred to control the pH including said fluctuation range to bewithin the above preferable pH range of 9 to 13.5.

Equipment which may be used as the disruption equipment 9, there may bementioned high-pressure homogenizer, sonication disruption machine,emulsification-dispersion machine, beadmill, which are mentioned above,and the like. In addition, two or more of the same or differentdisruption machines may be installed in parallel or in series. It ispreferable to use the above emulsification-dispersion machine orsonication disruption machine as the stirring means 2 for efficientlydiffusing the added alkali and for efficiently disrupting high-viscosityDNAs flowed out from the cell. In-line mixer type of these machines isalso manufactured, and those may function as both the pump 10 andstirring means 2 in FIG. 1, for example. In that case, the structureadvantageously becomes simple. In addition, general-purpose equipmentmay be used as the pH meter 7 and the pH detection and control equipment3.

Next, the step (b) in the present invention is a purifying method of aPHA which comprises treating with either an enzyme or a surfactant, orusing both in combination.

In the present invention, effects may be more remarkable as describedbelow by carrying out treatment of the step (b) to the PHA havingrelatively high purity obtained in the step (a).

It is generally considered that proteins, peptidoglycan (a cell wallcomponent), lipids, polysaccharides, nucleic acids and otherhydrocarbons are adhered to the PHA particles obtained in the step (a).The step (b) of the present invention is carried out for improving thepurity of a PHA by removing at least several of the above adherentcomponents.

The preferred practice of the present invention, a PHA separated in thestep (a) is used in the following step (b), not in a dried stateobtained by the separated PHA, but in such a state as suspended inwater, or as wetted with water after carrying out, e.g., centrifugationor membrane separation, for improving the treatment effect of the step(b).

When the treatment is carried out with an enzyme in the step (b), theenzyme to be used includes proteases, lipid degrading enzymes, cell walldegrading enzymes and DNases. As specific examples thereof, thefollowing enzymes may be listed. These may be used alone or two or moreof them may be used in combination.

-   (1) Proteases Alcalase, pepsine, trypsin, papain, chymotrypsin,    aminopeptidase, carboxypeptidase, etc.-   (2) Lipid Degrading Enzymes Lipases, phospholipases,    cholinesterases, phosphatases, etc.-   (3) Cell Wall Degrading Enzymes Lysozyme, amylase, cellulase,    maltase, saccharase, α-glycosydase, β-glycosydase, N-glycosydase,    etc.-   (4) DNases Ribonuclease, etc.

The enzymes to be used in this step are not restricted to the aboveones, but may include any enzymes provided that they are usable forindustrial products. Moreover, commercially available cleaning enzymedetergents, etc. may also be used.

Furthermore, it may be an enzyme composition containing e.g. astabilizing agent of enzymes or an anti-redeposition agent, togetherwith an enzyme, and is not restricted to a simple enzyme.

As the enzyme used for the purpose of decomposing and removing insolubleprotein and insoluble peptidoglycan adhering to the PHA, at least onespecies selected from proteases and cell wall decomposition enzymes arepreferred, and proteases are more preferred.

As a preferable protease, there may be mentioned, among those includedin the above exemplifications, Protease A, Protease P, Protease N(products of Amano Enzyme Inc.), Alkalase, Savinase, Everlase (productsof Novozymes Inc.), etc. as industrially applicable ones, and these aresuitable also in view of decomposition activity. Moreover, lysozyme orthe like is preferably used as the cell wall decomposition enzyme amongthose included in the above exemplification. But the enzymes are notlimited to these.

When carrying out an enzyme treatment, the treatment should be naturallycarried out at a temperature lower than the denaturation temperature ofthe enzyme. In many cases, the denaturation temperature of an enzyme islower than 65° C. Some enzymes have the denaturation temperature higherthan 65° C., and when using such enzymes, it is possible to carry outthe treatment at a temperature higher than 65° C. However, taking themolecular weight decrease of a PHA into consideration, the temperatureof the enzyme treatment is preferably 50° C. or less, and morepreferably 20° C. to 50° C.

The enzyme treatment is preferably continued until the treatmentproceeds to the required level, and it generally requires 0.5 to 2hours.

The amount of the enzyme to be used depends on the species and activitythereof. Although there is no particular restriction, but it ispreferably 0.001 to 10 parts by weight per 100 parts by weight of thepolymer, and more preferably 0.001 to 5 parts by weight or less in viewof cost.

The method of the present invention is advantageous as compared with aconventional method comprising treating the PHA-containing cell itselfwith an enzyme and disrupting the cell (see Japanese Kokoku PublicationHei-04-61638), in that a PHA may be produced in a low cost since only anamount of the enzyme to solubilize insolubilities slightly remaining inthe PHA is required.

In the step (b) of the present invention, it is possible to use asurfactant as a solubilizing agent for removing impurities adhering tothe PHA particles.

As the surfactant to be used in the present invention, there may bementioned an anionic surfactant, a cationic surfactant, an ampholyticsurfactant, a nonionic surfactant, or the like. These may be used aloneor two or more of them may be used in combination.

As the anionic surfactant, there may be mentioned an alkyl sulfate, analkyl benzene sulfonate, an alkyl or alkenyl sulfate, an alkyl oralkenyl ether sulfate, an α-olefin sulfonate, an α-sulfofatty acid saltor an ester thereof, an alkyl or alkenyl ether carboxylate, an aminoacid surfactant, an N-acyl amino acid surfactant, etc. Preferred amongthese are an alkyl sulfate having 12 to 14 carbon atoms in an alkylgroup, a straight chain alkyl benzene sulfonate having 12 to 16 carbonatoms in the alkyl group, and an alkyl sulfate or alkyl ether sulfatehaving 10 to 18 carbon atoms in the alkyl group. The counter ionpreferably includes, but not limited to, alkali metals such as sodiumand potassium, alkaline earth metals such as magnesium, alkanolaminessuch as monoethanolamine, diethanolamine and triethanolamine.

As the cationic surfactant, there may be mentioned an alkyltrimethylammonium salt, a dialkyldimethyl ammonium salt, etc.

As the ampholytic surfactant, there may be mentioned a carbobetainesurfactant, a sulfobetaione surfactant, etc.

As the nonionic surfactant, there may be mentioned a polyoxyalkylene(preferably oxyethylene) alkyl or alkenyl ether, a polyoxyalkylene(preferably oxyethylene) alkyl- or alkenylphenyl ether, apolyoxyethylene polyoxypropylene alkyl or alkenyl ether, polyoxyethylenepolyoxypropylene glycol, polyethyleneglycol, apolyoxyethylenealkylamine, a higher fatty acid alkanolamide, analkylglucoside, an alkylglucosamide, an alkylamine oxide, etc. Amongthese, preferred are those having high hydrophilicity and those with alow formation ability of liquid crystals, which is formed when admixedwith water, or those which generate no liquid crystals. Also use of apolyoxyalkyl ether having 10 to 14 carbon atoms and a polyoxyethylenealkyl ether having 10 to 14 carbon atoms, polyethylene glycol, etc. arepreferably used since they have comparatively preferablebiodegradability, but is not limited to these.

Specifically among the above-mentioned surfactants, preferred areanionic surfactants such as sodium dodecyl sulfate, sodiumdodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodiumoleate; and nonionic surfactants such as polyethylene glycol and apolyoxyethylene alkyl ether having 10 to 14 carbon atoms, etc. in viewof cost, amount to be used and effects produced by addition thereof. Twoor more of these may also be preferably used in combination.

The surfactants mentioned here in above are used in a generalcommercially available detergent, and an appropriate detergent forcleaning may be used as the surfactant.

In view of detergency, preferred are an anionic surfactant and anonionic surfactant. For the purpose of washing and removing proteinetc., it is preferable to use an anionic surfactant, and for the purposeof washing and removing fatty acid and oil, or when an enzyme is used incombination, a nonionic surfactant is preferably used. Furthermore, bothan anionic surfactant and a nonionic surfactant may be contained. Whenboth of them are contained, the weight ratio of the anionicsurfactant/the nonionic surfactant is preferably 1/100 to 100/10, morepreferably 5/100 to 100/20, still more preferably 5/100 to 100/100, andparticularly preferably 5/100 to 50/100.

The addition amount of the surfactant is not particularly restricted,but it is preferably 0.001 to 10 parts by weight per 100 parts by weightof a polymer, and more preferably it is 0.001 to 5 parts by weights inview of cost.

In addition, the treatment temperature in the surfactant treatment isnot particularly restricted, but preferably in the range of 20 to 50° C.in view of promoting solubirization of cell components other than a PHA.The treatment period is preferably 1 minute to 2 hours.

As a preferable embodiment of the present invention, there may bementioned a method comprising use of a surfactant with an enzymetreatment in combination because a higher purification effect may beproduced.

When an enzyme treatment and a surfactant are applied in combination,the amounts of the enzyme and the surfactant to be used are the same asthe above, respectively. The treatment temperature is preferably 20 to50° C., and the treatment period is preferably 1 minute to 2 hours.

The present inventors acknowledge the remarkable effects which areproduced when the two agents are used combinedly. The reason why theeffects are produced may be considered that a surfactant wouldefficiently remove a decomposed product which is released and becomesinsoluble by the enzyme decomposition, or that the structure of aprotein would be changed by a surfactant to be susceptible to an enzymedecomposition. In this case, the surfactant and the enzyme may beseparately prepared and appropriately admixed to use, but thecommercially available detergent containing an enzyme may be used as itis, since it is a mixture comprising a surfactant and an enzyme.

In the step (b) of the present invention, the enzyme or surfactanttreatment to be carried out may be freely selected according to reasonsor objects based on species of impurities to be removed, cost or otherrestrictions on the process, the purity of the objective PHA and/or thelike.

The enzyme treatment may be carried out in some divided steps. Forexample, one enzyme is used in the first step and subsequently the sameor different enzyme may be used in the following step. When one or morespecies of enzymes are used, it is convenient to treat a PHA in a singlestep using a mixture of enzymes if the contained enzymes do not digesteach other. Moreover, as mentioned above, the surfactant and enzymetreatment maybe carried out at the same time. Furthermore, it ispreferred to carry out both the enzyme treatment and the surfactanttreatment with stirring.

In the present invention, PHA particles obtained by the above-mentionedtreatment in the step (b) is washed with a hydrophilic solvent and/orwater for degreasing, deodorization and decolorization.

The hydrophilic solvent to be used in the step (b) is not particularlyrestricted, but specifically, there may be mentioned methanol, ethanol,acetone, acetonitrile, tetrahydrofuran, etc. Among these hydrophilicsolvents, methanol and ethanol are particularly preferred since they arecheap and have a good detergency.

In addition, the above hydrophilic solvents may be used as a mixturewith water. When using a mixed solvent composed of water and ahydrophilic solvent, the mixing volume ratio between water and thehydrophilic solvent (water/hydrophilic solvent) is preferably about 4/6to 0.5/9.5.

The amount of the above hydrophilic solvent used for washing is notparticularly restricted, but preferably not less than the amount equalto the polymer volume.

The temperature in washing is preferably not less than 20° C. but lessthan 60° C.

By washing a PHA with the above hydrophilic solvent and/or water, a PHAhaving more improved purity may be isolated.

In the present invention, it is possible to recover a PHA when the step(b) is completed, and the recovered PHA can be used as a material formolding and the like.

Since the PHA obtained in the step (b) is a fine microparticle havingthe particle diameter of as small as several microns, it is desirable toagglomerate the PHA to have a suitable particle diameter in the step(c), as described below, in view of separatability, handling property,etc.

The step (c) of the present invention comprises agglomerating PHAparticles by simple and convenient operations such as suspending the PHApurified in the step (b) in a hydrophilic solvent and/or water, andstirring said suspension at a temperature equal to or below the boilingpoint to enlarge the particle diameter.

The hydrophilic solvent to be used in the step (c) is not particularlyrestricted, but there may be mentioned, for example, alcohols such asmethanol, ethanol, 1-propanol, 2-propanol and butanol; ketones such asacetone and methylethylketone; ethers such as tetrahydrofuran anddioxane; nitriles such as a cetonitrile and propiononitrile; amides suchas dimethylformamide and acetoamide; dimethylsulfoxide, pyridine,piperidine and the like.

Preferred among them are methanol, ethanol, 1-propanol, 2-propanol,butanol, acetone, methylethylketone, tetrahydrofuran, dioxane,acetonitrile, propiononitrile, etc. in view of their removability. Morepreferred are methanol, ethanol, 1-propanol, 2-propanol, butanol,acetone, tetrahydrofuran, acetonitrile, etc. in view of their readyavailability.

Still more preferred is to use the solvent used for washing the PHA inthe step (b), since it becomes possible to reduce equipment cost and thelike because it becomes possible to proceed to the agglomeration processcontinuously, and/or because only one solvent tank is required.Therefore, as preferred solvents, there may be mentioned methanol,ethanol, acetone, acetonitrile, tetrahydrofuran, and the like. Amongthese, particularly preferred are methanol and ethanol since they arecheap and have good detergency.

Furthermore, the above hydrophilic solvent may be mixed with water to beused.

That is, the medium of a suspension may be any of a simple hydrophilicsolvent, simple water, or a mixed solvent comprising a hydrophilicsolvent and water. And preferred is the mixed solvent comprising thehydrophilic solvent and water. The concentration of the hydrophilicsolvent in the mixed solution is preferably 10 weight % or more and morepreferably 20 weight % or more in order to obtain more sufficientagglomerating effect. On the other hand, the upper limit of theconcentration of the hydrophilic solvent is 99 weight % or less,preferably 98 weight % or less, and more preferably 97 weight % or less.

The concentration of a PHA in the suspension of the step (c) is notparticularly restricted, but preferably 1 g/L or more, more preferably10 g/L or more, and still more preferably 30 g/L or more. The upperlimit is preferably 500 g/L or less, more preferably 300 g/L or less,and still more preferably 200 g/L or less in view of securing fluidityof the PHA suspension.

Stirring means in the step (c) of the present invention is notparticularly restricted and includes a stirring bath, etc. which causesturbulent flow.

The temperature on agglomeration in the step (c) of the presentinvention is preferably a room temperature (about 24° C.) or higher,more preferably 40° C. or higher, and still more preferably 60° C. orhigher. The upper limit is not particularly limited, and anytemperatures up to the boiling point of said suspension may be selected.

The step (c) maybe carried out under a condition of either normalpressure or high pressure. In the step (c) of the present invention, itis usually possible to cause agglomeration in such a very short time asabout several minutes, therefore there is no need to worry about themolecular weight decrease depending on a temperature when PHA isisolated soon after the agglomeration by filtration or the like.

By the agglomerating method of the step (c) according to the presentinvention, it becomes possible to enlarge the particle diameter of aPHA. For example, an agglomerate having the weight average diameter of10 μm or more, preferably 50 μm or more, and more preferably 100 μm ormore may be obtained. The upper limit is not particularly limited, butit is an agglomerate having the weight average diameter of 5000 μm orless, and preferably 3000 μm or less.

With increase of the particle diameter, recovery by filtration becomeseasy, and thus the equipment cost may be reduced in an industrialproduction. Herein, the method for filtration is not particularlyrestricted, but for example, a filter, a basket type separator, etc. maybe used.

To the PHA obtained by the present invention, coloring agents such aspigments and dyes, fillers such as inorganic or organic particles, glassfibers, whiskers and mica, stabilizing agents such as antioxidants andultraviolet absorbents, lubricants, mold-release agents,water-repellents, antibacterials, and other subsidiary additive agentsmay be added to prepare a PHA resin composition.

The said PHA resin composition may be formed into various forms, such asfibers, threads, ropes, textiles, fabrics, nonwoven fabrics, papers,films, sheets, tubes, boards, sticks, containers, bags, parts, foamedbodies, etc. Moreover, it may be also processed into a biaxial stretchedfilm. The formed products may be suitably used for such fields asagriculture, fishery, forestry, gardening, medical, sanitary products,clothing, non-clothing, packaging, and others. In particular, since thePHA obtained by the method of the present invention has quite highpurity, it is excellent in that it may be applied to fields requiringhigh purity, which a PHA obtained by the conventional methods cannot beapplied, for example, fields of film, medical, sanitary products, etc.

As mentioned above, by the recovery method of the present invention, itbecomes possible to efficiently recover a high-purity PHA from aPHA-containing microbial cell, which has been very difficult until now,and a PHA may be produced and provided at a low cost in a industrialscale.

By using the method for recovering a PHA comprising steps (a) and (b)according to the present invention, a polyhydroxyalkanoate isefficiently recovered in high purity from apolyhydroxyalkanoate-producing microbial cell, and is produced andprovided in a low cost on an industrial scale. And by further carryingout the step (c), it is possible to obtain an agglomerate of the PHAparticles.

BEST MODE FOR CARRYING OUT THE INVENTION

The following Examples illustrates the present invention in furtherdetail, but the invention is by no means limited to these.

The measuring method for each property is described below.

(Method to Measure 3HH mol %)

A PHA (PHBH) in a microbial cell after completion of culture wasrecovered by chloroform extraction and hexane crystallization, and wassubjected to analysis. The measurement of 3HH mol % was carried out bythe method described in Example 1 of Japanese Kokai Publication2001-340078. That is, PHBH was suspended in 2 ml of sulfuricacid-methanol mixed solution (15:85), chloroform (2 ml) was addedthereto, and the suspension was heated to 100° C. for 140 minutes. Aftercooling the suspension, 1 ml of distilled water was added and achloroform layer was recovered after stirring. This chloroform layer wassubjected to composition analysis using Shimadzu's gas chromatographGC-17A (NEUTRA BOND column produced by GL Science Inc.).

(Measuring Method for Residual Amount of Nitrogen in the PHA)

Just before the measurement, the recovered PHA (PHBH) was dried underreduced pressure at 50° C. for 5 hours, and the total nitrogen amountwas measured using trace nitrogen analyzer TN-10 (product of DiaInstruments Co., Ltd.). In the present invention, the measured nitrogenconcentration was converted to a corresponding protein concentration bymultiplying 6.38.

(Measuring Method for the Average Molecular Weight of the PHA)

After dissolving 10 mg of the recovered dried PHA in 5 ml of chloroform,insoluble matters were removed by filtration. Resultant solution wasanalyzed using Shimadzu's GPC system equipped with Shodex K805L (300×8mm, 2 columns-connected) (product of Showa Denko K. K.) with chloroformas a mobile phase. As the molecular weight standard sample, commerciallyavailable standard polystyrene was used. The molecular weight of a PHAin the microbial cell after completion of culture was measured in thesame manner as in the above method to measure 3HH mol %, that is, byrecovering a PHA from a PHA-containing microbial cell by chloroformextraction and hexane crystallization.

(Measurement of the Particle Diameter)

The average particle diameter of a PHA particle was measured by using amicrotrac particle diameter analyzer (product of NIKKISO Co., Ltd.,FRA). An aqueous suspension of the PHA was adjusted to the predeterminedconcentration, and the particle diameter corresponding to 50%accumulation amount of whole particles was determined as the averageparticle diameter.

EXAMPLE 1

(1) Step (a) Treatment

PHBH was produced by culturing Ralstonia eutropha obtained byintroducing a PHA synthase group gene derived from Aeromonas caviae(accession number FERM BP-6038) according to the method described inExample 1 of Japanese Kokai Publication 2001-340078. After completion ofthe culture, microbial cells were recovered by centrifugation to obtainan aqueous suspension containing 100 g/L of dried cells. The averagemolecular weight of PHBH in the recovered microbial cells was 1,400,000and 3HH composition was 6.8 mol %.

This aqueous suspension was subjected to a cell physical disruptionunder an alkali condition using the cell disruption equipment of FIG. 1.The cell disruption tank 11 was charged with 600 ml of aqueoussuspension of the PHA-containing microbial cell, and then the reactiontank was connected to high-pressure homogenizer model PA2K (disruptionequipment 9) manufactured by Italy Niro Soavi S.p.A, followed bycarrying out homogenization at a pressure of 600 to 700 kgf/cm². Bygradually adding 10% of sodium hydroxide after the lapse of 10 minutes,the cell aqueous suspension was adjusted to pH 12.5, and the suspensionwas circulated between the disruption tank 11 and the disruptionequipment 9 while maintaining this pH level. During this period, thetemperature of the cell disruption tank was maintained at 30° C. by athermoregulated circulation pump. Control of the pH level was carriedout as the followings; the pH electrode (pH indicator 7) was immersed inthe suspension in the cell disruption tank 11 and connected to LaboController MDL-6C manufactured by B. E. Marubishi Co., Ltd., andoperation parameters were set so that when the pH level of saidsuspension had dropped below a set value, a peristaltic pump (pump 4)would be actuated to deliver an aqueous solution of sodium hydroxideinto the suspension until the set value is attained. After 10time-circulations between the disruption tank 11 and the disruptionequipment 9, the suspension was centrifuged (9500 g, 30 minutes) toobtain a PHBH fraction. The obtained PHBH fraction was washed with watertwice, and was finally made into an aqueous suspension containing 100g/L of dried PHBH, and the aqueous suspension was used in the next step.

(2) Step (b) Treatment

The following tested agents were added to each 60 ml of PHBH suspensionobtained in the above (1). The addition amounts of the following testedagents are all indicated as weight % relative to the polymer weight in asuspension.

-   (1) 5 weight % of sodium dodecyl sulfate (SDS) (product of Wako Pure    Chemical Industries, Ltd.)-   (2) 0.08 weight % of Protease N (Product of Amano Enzyme Inc.)-   (3) 5 weight % of SDS and 0.08 weight % of Protease N-   (4) 5 weight % of SDS and 0.08 weight % of egg white lysozyme    (product of Wako Pure Chemical Industries, Ltd.)-   (5) 5 weight % of SDS, 0.08 weight % of Protease N and 0.08 weight %    of egg white lysozyme-   (6) 5 weight % of a synthetic detergent for cleaning (trade name:    Attack, product of Kao Corp.) (the amount is calculated to be such    an amount to contain an enzyme component of about 0.5 weight %)

Each of said suspension was stirred for 1 hour at 50° C. and pH of 7.0.Thereafter, PHBH was recovered by centrifugation, washed with 60 ml ofwater twice and with 60 ml of ethanol twice, and dried at 50° C. underreduced pressure to obtain a PHBH powder.

In addition, as a sample without the treatment of the step (b), a PHBHpowder obtained by washing the PHBH obtained in the above (1) withethanol twice and drying under reduced pressure was used. The resultsare shown in Table 1. TABLE 1 PHA Total amount of Total amount of puritySample nitrogen μg/g protein mg/g (%) Without treatment 5500 35.09 96.49{circle around (1)} SDS 600 3.83 99.62 {circle around (2)} Protease N540 3.45 99.66 {circle around (3)} SDS + Protease N 130 0.83 99.92{circle around (4)} SDS + Lysozyme 69 0.44 99.96 {circle around (5)}SDS + Protease N + 110 0.70 99.93    Lysozyme {circle around (6)}Synthetic powdered 190 1.21 99.88    detergent

PHBH showed purity of 99.5% or more in the step (b), and was conformedto have an effect as compared with that conducted no treatment. Althoughuse of SDS alone was effective, the purity was further improved by usingan enzyme in combination. Moreover, the commercially available syntheticdetergent is considered to be preferable since it also has a good effectand is cheap.

EXAMPLE 2 Carrying out the Recovery Process in a Total Flow (Steps (a)Through (c))

Ralstonia eutropha cultured in the same manner as in Example 1(1) wasrecovered by centrifugation. This cell was suspended in water to preparean aqueous suspension containing 100 g/L of dried cells. The averagemolecular weight of PHBH in the recovered cells was about 1,470,000 and3 HH composition was 5.1 mol %. Using 400 ml of this suspension, ahigh-pressure disruption according to the method described in Example1(1) was carried out while maintaining pH at about 12.5. Aftercompletion of the treatment, a PHBH fraction was recovered bycentrifugation, and washed with water twice.

The obtained PHBH fraction was suspended in water to prepare an aqueoussuspension containing 100 g/L of dried cells. To this suspension, 0.2weight % of Protease N, 0.2 weight % of lysozyme and 4 weight % of SDSrelative to the polymer weight were added, and stirred at 50° C. and pHof 7.0 for 1 hour. After completion of the treatment, PHBH was washedwith water twice.

The obtained PHBH fraction was suspended in water to prepare an aqueoussuspension with a concentration of 200 g/L. 290 ml of 95% ethanol wasadded and suspended in said suspension, and successively PHBH wasprecipitated by centrifugation. 290 ml of the supernatant was removed,and 290 ml of 95% ethanol was added again to the polymer fraction tosuspend PHBH. This ethanol washing was carried out twice, and asuspension added with 290 ml of 95% ethanol was prepared. Said PHBHsuspension was gradually added to 290 ml of ethanol at 70° C. in 15minutes, and PHBH was agglomerated by further stirring for 10 minutesafter completion of addition. The agglomerated PHBH was recovered byfiltration using Kiriyama filter paper (No. 58) (product of KiriyamaGlass Works Co.). PHBH on the filter paper was washed with 120 ml (equalamount to PHBH content) of 95% ethanol twice. The obtained PHBH wasdried in vacuum at 50° C. The results of the PHBH purity analysis areshown in Table 2. TABLE 2 Total Total amount of amount of Particlenitrogen protein Purity size Molecular PHBH μg/g mg/g (%) μm weight×10⁻⁶ After — — — 7.5 1.47 step (b) After 140 0.89 99.91 203 1.42 step(c)

As the result, 56 g of PHBH having the purity of 99.91% was obtained(recovery percentage from the material before the step (a) is 93%). Theaverage molecular weight after the step (c) was 1,420,000, which meantthat decrease was only 3.4% from the molecular weight from the materialbefore the step (a).

POSSIBILITY OF INDUSTRIAL APPLICATION

Using the method for recovering a PHA comprising the steps (a) and (b)of the present invention, it becomes possible to efficiently recover ahigh-purity PHA from a PHA-containing microbial cell, and PHA may beproduced and provided at a low cost in an industrial scale. Moreover, byusing the step (c) agglomerated PHA particles may be obtained.

1. A method for recovering a polyhydroxyalkanoate from apolyhydroxyalkanoate-containing microbial cell which comprises thefollowing steps (a) and (b); (a) a step comprising adding an alkali toan aqueous suspension of the polyhydroxyalkanoate-containing microbialcell while stirring and carrying out a physical disruption treatment todisrupt the cell, solubilizing or emulsifying cell substances other thanthe polyhydroxyalkanoate in said cell, and then separating thepolyhydroxyalkanoate from the aqueous suspension, and (b) a stepcomprising treating the separated polyhydroxyalkanoate with an enzymeand/or a surfactant to solubilize impurities adhering to thepolyhydroxyalkanoate or to solubilize them after decomposing, and thenwashing the polyhydroxyalkanoate with a hydrophilic solvent and/orwater.
 2. The method for recovering a polyhydroxyalkanoate according toclaim 1 which further comprises the following step (c); (c) a stepcomprising suspending the washed polyhydroxyalkanoate in a hydrophilicsolvent and/or water and stirring at a temperature equal to or below theboiling point of said suspension and agglomerating thepolyhydroxyalkanoate to enlarge the particle diameter thereof, and thenseparating the agglomerated polyhydroxyalkanoate from the suspension. 3.The method for recovering a polyhydroxyalkanoate according to claim 1,wherein the polyhydroxyalkanoate is a copolymer obtainable bycopolymerizing at least two species of hydroxyalkanoate monomersselected from the group consisting of 3-hydroxybutyrate,3-hydroxyvalerate, 3-hydroxypropionate, 4-hydroxybutyrate,4-hydroxyvalerate, 5-hydroxyvalerate, 3-hydroxypentanoate,3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate,3-hydroxynonanoate and 3-hydroxydecanoate.
 4. The method for recoveringa polyhydroxyalkanoate according to claim 3, wherein thepolyhydroxyalkanoate is a copolymer composed of 3-hydroxyhexanoate andat least one species among said hydroxyalkanoate monomers other than3-hydroxyhexanoate.
 5. The method for recovering a polyhydroxyalkanoateaccording to claim 4, wherein the polyhydroxyalkanoate is a copolymercomposed of 3-hydroxyhexanoate and 3-hydroxybutyrate.
 6. The method forrecovering a polyhydroxyalkanoate according to claim 1, wherein, in thestep (a), the physical disruption treatment is carried out by ahigh-pressure homogenizer.
 7. The method for recovering apolyhydroxyalkanoate according to claim 1, wherein, in the step (a), thealkali is added continuously or intermittently while controlling a pHlevel.
 8. The method for recovering a polyhydroxyalkanoate according toclaim 7, wherein, in the step (a), the pH level is controlled between 9and 13.5.
 9. The method for recovering a polyhydroxyalkanoate accordingto claim 1, wherein the alkali to be used in the step (a) is at leastone species selected from the group consisting of sodium hydroxide,potassium hydroxide, lithium hydroxide and sodium carbonate.
 10. Themethod for recovering a polyhydroxyalkanoate according to claim 1,wherein the enzyme to be used in the step (b) is at least one speciesselected from the group consisting of proteases, lipid degradingenzymes, cell wall degrading enzymes and DNases.
 11. The method forrecovering a polyhydroxyalkanoate according to claim 1, wherein thesurfactant to be used in the step (b) is at least one species selectedfrom the group consisting of anionic surfactants, cationic surfactants,ampholytic surfactants and nonionic surfactants.
 12. The method forrecovering a polyhydroxyalkanoate according to claim 1, wherein thehydrophilic solvent to be used for the washing in the step (b) is atleast one species selected from the group consisting of methanol,ethanol, acetone, acetonitrile and tetrahydrofuran.
 13. The method forrecovering a polyhydroxyalkanoate according to claim 2, wherein thehydrophilic solvent used in the step (c) is at least one speciesselected from the group consisting of methanol, ethanol, acetone,acetonitrile and tetrahydrofuran.
 14. The method for recovering apolyhydroxyalkanoate according to claim 1, wherein a microorganismcontaining the polyhydroxyalkanoate is a microorganism selected from thegroup consisting of species belonging to the genus Aeromonas,Alcaligenes, Azotobacter, Bacillus, Clostridium, Halobacterium,Nocardia, Rhodospirillum, Psuedomonas, Ralstonia, Zoogloea, Escherichia,Candida, Saccharomyces and Yarrowia.
 15. The method for recovering apolyhydroxyalkanoate according to claim 14, wherein the microorganismcontaining the polyhydroxyalkanoate is Aeromonas caviae.
 16. The methodfor recovering a polyhydroxyalkanoate according to claim 1, wherein themicroorganism containing the polyhydroxyalkanoate is a transformantobtainable by introducing a polyhydroxyalkanoate synthase group genederived from Aeromonas caviae.
 17. The method for recovering apolyhydroxyalkanoate according to claim 16, wherein the microorganismcontaining the polyhydroxyalkanoate is Ralstonia eutropha obtainable byintroducing a polyhydroxyalkanoate synthase group gene derived fromAeromonas caviae.
 18. The method for recovering a polyhydroxyalkanoateaccording to claim 2, wherein the polyhydroxyalkanoate is a copolymerobtainable by copolymerizing at least two species of hydroxyalkanoatemonomers selected from the group consisting of 3-hydroxybutyrate,3-hydroxyvalerate, 3-hydroxypropionate, 4-hydroxybutyrate,4-hydroxyvalerate, 5-hydroxyvalerate, 3-hydroxypentanoate,3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate,3-hydroxynonanoate and 3-hydroxydecanoate.
 19. The method for recoveringa polyhydroxyalkanoate according to claim 18, wherein thepolyhydroxyalkanoate is a copolymer composed of 3-hydroxyhexanoate andat least one species among said hydroxyalkanoate monomers other than3-hydroxyhexanoate.
 20. The method for recovering a polyhydroxyalkanoateaccording to claim 2, wherein, in the step (a), the physical disruptiontreatment is carried out by a high-pressure homogenizer.